4-Aminopyridyl-based CYP51 inhibitors as anti-Trypanosoma cruzi drug leads with improved pharmacokinetic profile and in vivo potency

Article abstract of DOI:10.1021/jm500448u

CYP51 is a P450 enzyme involved in the biosynthesis of the sterol components of eukaryotic cell membranes. CYP51 inhibitors have been developed to treat infections caused by fungi, and more recently the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. To specifically optimize drug candidates for T. cruzi CYP51 (TcCYP51), we explored the structure-activity relationship (SAR) of a N-indolyl-oxopyridinyl-4- aminopropanyl-based scaffold originally identified in a target-based screen. This scaffold evolved via medicinal chemistry to yield orally bioavailable leads with potent anti-T. cruzi activity in vivo. Using an animal model of infection with a transgenic T. cruzi Y luc strain expressing firefly luciferase, we prioritized the biaryl and N-arylpiperazine analogues by oral bioavailability and potency. The drug-target complexes for both scaffold variants were characterized by X-ray structure analysis. Optimization of both binding mode and pharmacokinetic properties of these compounds led to potent inhibitors against experimental T. cruzi infection.

Full text of DOI:10.1021/jm500448u

Article
pubs.acs.org/jmc
Open Access on 08/07/2015
4
‑Aminopyridyl-Based CYP51 Inhibitors as Anti-Trypanosoma cruzi
Drug Leads with Improved Pharmacokinetic Profile and in Vivo
Potency
Claudia M. Calvet,†,‡,⊥ Debora F. Vieira, Jun Yong Choi, Danielle Kellar, Michael D. Cameron,
†,‡
#
†,‡
∇
†
,‡
†,‡,§
∥
∇
∇
Jair Lage Siqueira-Neto, Jiri Gut,
Jonathan B. Johnston, Li Lin, Susan Khan,
James H. McKerrow,†,‡ William R. Roush,* and Larissa M. Podust*
,#
,†,‡
†
‡
§
∥
Center for Discovery and Innovation in Parasitic Diseases, Department of Pathology and Department of Medicine, Department of
Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94158, United States
⊥
Cellular Ultra-Structure Laboratory, Oswaldo Cruz Institute (IOC), FIOCRUZ, Rio de Janeiro, Re de Janeiro 21040-362, Brazil
#
∇
Department of Chemistry, Department of Molecular Therapeutics, Scripps Florida, Jupiter, Florida 33458, United States
*
S Supporting Information
ABSTRACT: CYP51 is a P450 enzyme involved in the
biosynthesis of the sterol components of eukaryotic cell
membranes. CYP51 inhibitors have been developed to treat
infections caused by fungi, and more recently the protozoan
parasite Trypanosoma cruzi, the causative agent of Chagas
disease. To specifically optimize drug candidates for T. cruzi
CYP51 (TcCYP51), we explored the structure−activity
relationship (SAR) of a N-indolyl-oxopyridinyl-4-aminopro-
panyl-based scaffold originally identified in a target-based
screen. This scaffold evolved via medicinal chemistry to yield
orally bioavailable leads with potent anti-T. cruzi activity in
vivo. Using an animal model of infection with a transgenic T.
cruzi Y luc strain expressing firefly luciferase, we prioritized the
biaryl and N-arylpiperazine analogues by oral bioavailability and potency. The drug−target complexes for both scaffold variants
were characterized by X-ray structure analysis. Optimization of both binding mode and pharmacokinetic properties of these
compounds led to potent inhibitors against experimental T. cruzi infection.
INTRODUCTION
treatment of T. cruzi infection in humans and animals,
identified in drug discovery efforts in academic and product
development partnerships, is sterol 14-demethylase, CYP51.
■
Chagas disease is caused by infection of mammalian host cells
by the parasitic protozoon Trypanosoma cruzi, usually trans-
mitted by blood-sucking bugs of the subfamily Triatominae. T.
8
1
CYP51 is a clinically validated therapeutic target of antifungal
9
azole drugs of the “conazole” pedigree. Their efficacy against a
cruzi colonizes the heart, gastrointestinal tract, and nervous
system, causing progressive inflammation which can lead to
chronic human cardiopathy and/or gastrointestinal dysfunc-
broad spectrum of fungal pathogens have defined azole
compounds as an indispensable tool for controlling fungal
10,11
12
2
3
diseases in humans
and crop plants. The replicative
tion. According to the World Health Organization, T. cruzi
infects around 7−8 million people worldwide, mostly in Latin
America. More recently Chagas disease has expanded beyond
tropical and subtropical zones to become an emerging
economic and health burden in the United States, Spain, and
intracellular amastigote form of T. cruzi is particularly sensitive
13
to CYP51 inhibitors due to its obligate reliance on de novo
synthesis of sterols as membrane building blocks. Posaconazole
(
Noxafil, Merck), the most recent member of the family to be
1
4,15
4
marketed,
demonstrates excellent anti-T. cruzi potency,
the UK. Antiparasitic treatment is recommended for all acute
high percentages of parasitological cure in animal mod-
and chronic patients, including those with the indeterminate
chronic form of Chagas disease.
chemotherapeutic options for Chagas disease are limited to
benznidazole and nifurtimox, which are used during the acute
phase but may cause severe gastrointestinal, dermatological,
1
3,16−18
5
,6
els,
and has a precedent of successfully curing a patient
At the same time,
with chronic Chagas disease and systemic lupus erythemato-
1
9,20
sus.
Posaconazole’s efficacy against model T. cruzi infection
is attributed to its favorable DMPK properties, including oral
7
and neurological side effects. The efficacy of these drugs in the
chronic stage is debated, and neither drug is approved by the
FDA for use in the United States. A promising target for
Received: March 21, 2014
©
XXXX American Chemical Society
A
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a
Scheme 1. Syntheses of Compounds 1−12
a
Reagents and conditions: (a) arylboronic acid, 5 mol % Pd (dba) , 10 mol % PCy , 2M K PO , dioxane, 100 °C (microwave), 1 h, ca. 90%; (b)
2
3
3
3
4
H SO /MeOH (1/10), 70 °C, 24 h, 91%; (c) H (balloon), Pd/C, MeOH−acetone, 23 °C, 24 h, 92%; (d) 4-fluorobenzyl bromide, K CO , acetone,
2
4
2
2
3
7
0 °C, 5 h, 95%; (e) 10% NaOH (aq), MeOH/THF (1/1), 60 °C, 3 h, ca. 95%; (f) acetic anhydride, Et N, CH Cl , 0−23 °C, 1 h, 84%; (g) 10%
3
2
2
NaOH (aq), MeOH/THF (1/1), 23 °C, 2 h, 36%; (h) 1-(aryl)piperazine, Pd(OAc) , P(o-tolyl) , Cs CO , toluene, 60 °C, 48 h, ca. 70%; (i) 13, 14,
1
2
3
2
3
5, 16, 17, 18, 19, 22b, 24b, 26b, 27b, 28b, or 29b (as appropriate), PyBOP, HOBt, Et N, CH Cl , 23 °C, 1 h, ca. 70%.
3 2 2
availability, long terminal half-life, and large volume of
candidate and a nontoxic herbicide, respectively, have been
optimized to specifically target T. cruzi. Also, recently entering
the anti-Chagas pipeline are azole derivatives from a collection
2
1,22
distribution.
Nevertheless, the majority of azole inhibitors
in clinical use or development are not powerful enough to
40
eradicate T. cruzi from human patients or experimental
of the Novartis Research Institute (Vienna, Austria).
2
3−25
animals.
Our collaboration has focused on de novo structure-aided
hit-to-lead optimization of a pyridine-based molecular scaffold
discovered by a Mycobacterium tuberculosis CYP51-based
The similarity between sterols and their biosynthetic
pathways in fungi and protozoa has led to several clinical trials
of azole antifungal agents for treating Chagas disease patients.
41−43
screen.
Structure-aided chemical tailoring, guided by a T.
26
Posaconazole and ravuconazole (Eisai Co, Tokyo, Japan),
both acquired from industry antifungal programs, have been
cruzi whole cell-based assay, has identified the favorable R-
configuration of the carbon chiral center and enabled
exploration of structural features contributing to enhanced
microsomal stability and diminished inhibitory potential toward
20
advanced into clinical trials. The results thus far have been
less than optimal, with only temporary efficacy over a longer
time frame, indicating the need for further trials at different
4
4,45
a panel of mammalian drug-metabolizing CYP enzymes.
As
2
7,28
doses or in combination with benznidazole.
Neither drug
a result of these earlier medicinal chemistry efforts, the EC of
5
0
has yet been proven superior to benznidazole in treating T.
cruzi infection in patients, indicating that the quest for a cure
for Chagas disease must continue. It is notable that neither
posaconazole nor ravuconazole were initially developed to
target T. cruzi CYP51. Two next-generation promising CYP51
the new scaffold improved up to 4 orders of magnitude
compared to that of the parental compound, LP10.
We have now used a mouse model of acute T. cruzi infection
to evaluate the potency and oral bioavailability of optimized
CYP51 inhibitors. We are focusing on two biaryl and N-
arylpiperazine scaffold variants, so named based on the
4
5,46
29−36
37−39
inhibitors, tipifarnib
and fenarimol,
a cancer drug
B
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structure of the longest substituent at the chiral carbon center.
Using X-ray crystallography, we have characterized the binding
modes of three analogues, at least one from each scaffold group,
to resolutions of 2.04, 2.43, and 2.80 Å. These structures can
now guide site-directed medicinal chemistry interventions
aimed at further lead improvements. The structural differences
between scaffolds affect the binding mode and pharmacokinetic
behavior of these compounds. Using a four-day dosing regimen,
we have prioritized inhibitors by efficacy in the animal model
and correlated them with efficacy in the cell-based assay used to
rank compounds for animal studies. Small differences in
chemical structure dramatically affect the compounds’ binding
modes, oral availability, terminal half-lives, and efficacy against
T. cruzi. Five CYP51 inhibitors presented in this work
suppressed >97% of parasite load in mice when administered
orally at 50 mg/kg, bid, for four consecutive days.
RESULTS
■
Chemistry. Syntheses of compounds 1−12 which were
prepared for this study are summarized in Scheme 1. Palladium-
mediated coupling reactions of 4-bromo-2-fluorobenzoic acid
and methyl 4-bromo-2-fluorobenzoate (25) with various aryl
boronic acids and N-aryl piperazines served as key steps of the
syntheses of intermediates 13−19 and 26a−29a, respec-
4
4,45
tively.
The coupling reactions of 4-bromo-2-fluorobenzoic
acid with aryl boronic acids, performed under microwave
irradiation (100 °C for 1 h, or 120 °C for 2 h for the sterically
hindered products such as 13 and 16), provided intermediates
Figure 1. Animal model of T. cruzi Y luc infection. (A) Development
of parasitemia in the untreated mice over the course of 21 days
postinfection with T. cruzi Y luc parasites. Parasite count in fresh blood
samples (red) was paralleled by the luminescence reading of whole
amimals (blue). Each measurement is an average of five mice. (B)
Evolution of the parasitemia in a single experimental animal by
luminescence.
13−19 in ca. 90% yields. Intermediates 26a, 27a, 28a, and 29a
were prepared by the coupling of N-arylpiperazine derivatives
with methyl 4-bromo-2-fluorobenzoate (25) at 60 °C in
toluene, which were then hydrolyzed under basic conditions to
provide carboxyl intermediates 26b, 27b, 28b, and 29b.
Conversion of 19 to methyl ester 20 set the stage for
replacement of the benzyl group with a 4-fluorobenzyl unit
through catalytic hydrogenation (Pd/C and H ) and then O-
alkylation with 4-fluorobenzyl bromide. N-Acetylation of 23
produced 24a, which was hydrolyzed to carboxylic acid 24b. All
disseminates throughout the body and mice develop heavy
parasitemia in the following 5−11 days (Figure 1B). Because
trypanocidal activity of sterol biosynthesis inhibitors is time-
dependent and results from complete depletion of the
ergosterol pool within 72−96 h of drug exposure in
2
44
carboxylic acid intermediates were coupled with D-tryptophan
4
5
derivative 30 to provide the final compounds 1−12.
1
3,48,49
Animal Model. To rapidly assess the effect of drugs on the
parasite load in vivo, a four-day dosing mouse model utilizing
transgenic T. cruzi Y luc strain expressing firefly luciferase
vitro,
the four-day in vivo regimen is justifiable. To
assess the efficacy of test compounds, mice were infected with
T. cruzi Y luc strain for three days. Starting on day 4, the
infected mice were treated with test compounds either via
intraperitoneal injection (ip) or oral gavage (po) for four
consecutive days, twice a day (bid). At day 7 postinfection, the
T. cruzi luminescent signal in the mice was read upon injection
of D-luciferin.
4
7
reporter gene was adopted. The use of the luciferase-
transfected strain enables immediate and direct reading of
luminescence by imaging live animals on the detector plate.
Given that the luciferase reporter carried by T. cruzi Y luc strain
sustained only a limited number of passages in mammalian cell
culture, we first tested the longevity of this marker in the mouse
model by correlating luminescence in whole animal with
parasite count in blood. The experiment was conducted in a
Anti-T. cruzi Activity by ip Administration. In vivo
activity of inhibitors, ranked a priori by EC in the cell-based
5
0
44−46
assay previously described,
was assessed by intraperitoneal
5
group of five mice, each infected with 10 trypomastigotes. The
injection in the first two independent experiments (Figure
2A,B), when the mice were treated with 40 mg/kg of test
compounds, ip, bid. In the subsequent experiments, com-
pounds were administered orally. As shown in Figure 2, the
parasite load in the untreated animals significantly increased
over a seven-day period. Posaconazole used as a positive
control produced 94−99% inhibition of parasitemia. The
parental hit, LP10, showed little efficacy under this treatment
regimen, while the analogues 3, 4, and 8 suppressed parasite
load by ≥97% over the four-day treatment period with a
statistically significant difference (p ≤ 0.05) compared to the
vehicle-treated controls. The efficacy of R-2 (52%), 1 (35%), 2
impact of the transgenic T. cruzi Y luc strain was not lethal. All
mice survived untreated 30 days postinfection, during which
parasitemia was monitored for 21 days in live animals both by
the luciferase reaction and parasite count by direct microscopic
examination of fresh blood smears. The parasite count in blood
dropped below the detectable level on day 21, while the
luciferase signal persisted through day 21 (Figure 1A). All mice
on day 21 appeared normal and agile.
On the basis of this experiment, a four-day dosing regimen
was constructed with drug administration beginning on day 3,
after infection is established. If left untreated, infection quickly
C
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isolated from mouse embryos (Table 2). The biaryl compounds
5
, 6, and 7 inhibited proliferation and viability of mammalian
cells at concentrations below 90 μM, while N-arylpiperazine
compounds 11 and 12 did not affect or only barely affected
mammalian cells at 90 μM, the highest concentration applied in
this experiment. Cardiomyocytes and hepatocytes were equally
susceptible to compound action, with EC50 values in the same
range for each of the tested compounds. The large difference in
selectivity index (SI), calculated as the ratio between the EC50
values for inhibition of host cells and T. cruzi intracellular
amastigotes, was due to a wider range of compound efficacy
against T. cruzi. On the selectivity scale, compound 6 was
ranked the lowest (<200) and compound 11 the highest
(
>5000). The three most selective compounds, 5, 11, and 12,
belong to different scaffold variants (Table 2).
The anti-T. cruzi EC50 values for the new biaryl analogues,
although still effective in the nanomolar range, increased
compared to the immediate precursors R-2, 1, and 2 (Table 1).
The N-arylpiperazine analogues retained the potency of their
immediate precursors. Among the six new analogues, those
belonging to the biaryl scaffold variant had a notably longer
half-life in a microsome stability assay and less inhibition of
human CYPs in vitro (Table 1). Inhibition of four major drug-
metabolizing human CYP enzymes, 1A2, 2C9, 2D6, and 3A4,
contributing to the oxidative metabolism of vast majority of
50
drugs in clinical use, has been assessed. With the exception of
Figure 2. Efficacy of compounds upon ip administration. In two
independent experiments, (A) and (B), compounds were administered
at 40 mg/kg, ip, bid. Luminescence in mice was read upon luciferin
injection on day 3 postinfection and prior to treatment (black bars)
and on day 7 postinfection and after four days of treatment (gray
bars). Each data point is an average of five mice; dpi-days
postinfection. Posaconazole (Pos) served as a positive control. Percent
inhibition for each compound is calculated relative to the untreated
control on day 7 postinfection. *Values significantly different than
vehicle-treated controls (p ≤ 0.05), except for LP10.
2
C9, which was inhibited 90−96% by the majority of the tested
compounds regardless of scaffold, inhibition of three other
human CYPs by biaryl inhibitors dropped below 30%. At the
same time, N-arylpiperazine compounds inhibited 3A4 by 85−
9
9
0% and 2D6 by 14−87%. Inhibition of 1A2 dropped below
% for both scaffold variants with an exception of 11 (20% 1A2
inhibition). For comparison, ketoconazole and posaconazole at
1 μM concentration inhibited 22/22/4/95 and −11/−3/−9/
68% of human 1A2/2C9/2D6/3A4 enzymes, respectively.
Thus, both ketoconazole and posaconazole hit 3A4, while other
CYPs were only modestly inhibited or not inhibited at all under
the experimental conditions used in these studies.
(
84%), and 9 (52%) was attenuated relative to the three most
effective inhibitors. The outcomes of R-2, 1, and 2 were
particularly unexpected, as these compounds had superior EC50
in the T. cruzi cell-based assay (Table 1). The effect of
compounds on the host cells was monitored using a high-
content image-based whole cell assay used to prioritize
inhibitors for testing in the animal model. No toxicity toward
mouse myoblasts used to harbor parasites has been observed
for any compound featured in Table 1 up to 10 μM, the highest
concentration used to generate dose−response curves
To confirm the mechanism of action, two newly synthesized
compounds, 3 and 12, one for each scaffold variant, have been
tested for disruption of sterol biosynthesis pathway in
intracellular T. cruzi amastigotes. The assay was conducted as
51
described elsewhere. Lipids extracted from intracellular
amastigotes were analyzed by gas chromatography and mass
spectrometry (GC-MS) and identified as described previ-
52
ously. DMSO and benznidazole were used as negative
controls; posaconazole served as positive control. Cholesterol
was the only peak originated from the host cells (Supporting
Information, Figure S2). The major sterol observed in
untreated amastigotes was episterol (peak d), followed by
approximately equal amounts of fecosterol (e) and cholesta-
7,24-dien-3β-ol (peak a). As a result of treatment, two 14-
methylated precursors, lanosterol (f) and eburicol (h), were
prominent in the GC-MS traces of the CYP51 inhibitors
posaconazole and compounds 3 and 12 with concomitant
decline in episterol, fecosterol, and other 14-demethylated
intermediates. No changes in lipid composition have been
observed in benznidazole-treated samples compared to DMSO.
Benznidazole is the drug currently used to treat Chagas disease
and does not target ergosterol synthesis pathway, inducing the
(
Supporting Information, Figure S1)
The most active compounds following intraperitoneal
adminstration, 3, 4, and 8, belonged to distinct scaffold
variants, referred to as biaryl and N-arylpiperazine (Table 1).
Both scaffolds retain the invariant N-indolyl-oxopyridinyl-4-
aminopropanyl portion of the skeleton while carrying different
side chain substituents at the chiral carbon center in the R-
configuration: a biaryl moiety or an N-arylpiperazine moiety, in
which the piperazine ring separates two aromatic rings of the
biaryl structure, thus conferring flexibility to the otherwise rigid
system.
Next Generation of Compounds. Six follow-up ana-
logues, 5, 6, 7 (biaryl scaffold variant), 10, 11, and 12 (N-
arylpiperazine scaffold variant), differed within each group by
the halogenation pattern of the terminal phenyl ring (Table 1).
Cytotoxicity of five of these new analogues was assessed against
two different cell types, cardiomyocytes and hepatocytes,
53
formation of free radicals in the parasite. On the basis of this
experiment, we conclude that in the course of hit-to-lead
optimization compounds of the N-indolyl-oxopyridinyl-4-
D
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j
Table 1. Summary of the in Vitro and in Vivo Properties of CYP51 Inhibitors
a
b
c
Each measurement performed in triplicate. apparent KD values (see Supporting Information). Each measurement is an average of five mice
d
treated 40 mg/kg (60% DMSO), ip, bid, for 4 days. Each measurement is an average of five mice treated 25 mg/kg (20% solutol), po, bid, for 4
days. Each measurement is an average of five mice treated 50 mg/kg (20% solutol), po, bid, for 4 days. Stability of compounds in human (h), rat
e
f
g
(
r), and mouse (m) liver microsomes as evaluated compared to the Sunitinib reference (see Supporting Information). Inhibition of CYPs as
h
evaluated in human liver microsomes using selective marker substrates for each CYP (see Supporting Information). LP10 was previously
52
characterized in vivo and close LP10 analogues were assessed for stability (4−7 min in both human and mouse liver microsomes) and cross-
44 i
reactivity (21−23% inhibition of 1A2; 92% for 2C9; 62−77% for 2D6; and 75−55% inhibition of 3A4) previously. Compound R-2 was previously
45 j
characterized structurally and in vitro.
N/D: not determined.
Table 2. Cytotoxicity of Compounds in Mouse Hepatocytes
and Cardiomyocytes
establish optimal formulation for the bioavailability test, both 2-
54
hydroxypropyl-β-cyclodextrin (HPβCD) and the nonionic
55
surfactant Kolliphor (a mixture of polyoxyethylene esters of
hepatocytes
selectivity index
cardiomyocytes
1
2-hydroxystearic acid and free polyethylene glycol, also known
selectivity index
as solutol) were tested as vehicles for oral administration of
compounds. The plasma concentration−time curves and the
tissue distribution of compounds following oral administration
of a single 50 mg/kg dose in 20% Kolliphor or 20% HPβCD
demonstrated remarkable differences between two vehicles
a
compd EC50 (μM)
(SI)
EC50 (μM)
(SI)
b
5
6
7
1
1
68.1 ± 7.2
28.3 ± 4.1
48.1 ± 12.5
>90
3243
166
53.9 ± 8.1
22.2 ± 2.3
23.3 ± 5.9
>90
2568
131
616
299
1
2
>5000
>3000
>5000
2869
(
Table 3). The PK parameters extracted from the concen-
>90
83.2 ± 4.7
tration−time curves showed higher oral bioavailability for the
majority of compounds in Kolliphor compared to that in
HPβCD. A notable exception was compound 12, which was
comparable in both vehicles. When administered in Kolliphor,
compound 7 demonstrated superior half-life (16.8 h), followed
by 11 (4.6 h) and 12 (4.4 h). The shortest half-life was
observed for 6 and 2 (<3.3 h). The highest Cmax and AUC
values and the lowest clearance were achieved by 5 and 7. The
lowest Cmax and highest clearance were observed for 11 and 2.
The majority of tested compounds had in vivo half-lives
necessitating twice a day administration.
a
Calculated as a ratio between the host cell and intracellular T. cruzi
amastigote survival, both expressed as the corresponding EC50 values.
b
Each measurement performed in duplicate.
aminopropanyl-based SAR series retained the mechanism of
action and specifically target T. cruzi CYP51.
Pharmacokinetic Behavior: Cyclodextrin versus Kolli-
phor. Most T. cruzi parasites in chronic infection reside
intracellularly, largely in heart, gut, and skeletal muscles;
therefore, anti-T. cruzi drugs must be lipophilic enough to
penetrate cell membranes and deep tissues. On the other hand,
poor water solubility leads to ineffective absorption. Aiming to
Tissue Tropism. Tissue tropism, an important factor
defining efficacy of drugs against T. cruzi, was assessed 2 and
E
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Table 3. PK Parameters in HPβCD and Kolliphor
a
Each measurement is an average of three mice received a single 50 mg/kg oral dose of test compound as 10 mg/mL suspension in 20% HPβCD
gray fields) or 20% Kolliphor (white fields).
(
8
h postexposure following oral administration of compounds
(Figure 4A). To establish a po administration dose, dose−
response curves were obtained for four inhibitors: 5, 6, 7, and
12 (Figure 4B). The highest dose of 50 mg/kg bid was the
most efficient for all the compounds, while 25 mg/kg bid dose
allowed us to efficiently discriminate among and prioritize
inhibitors. No signs of toxicity (weight loss, mobility issues,
appearance) were observed in the four-day studies.
in Kolliphor or HPβCD at 50 mg/kg. Tabulated data are
included in the Supporting Information (see Tables S1 and S2),
while a graphic summary is provided in Figure 3. Tissue
exposure was enhanced for a majority of the compounds when
administered in Kolliphor versus HPβCD, again with the
exception of 12, which was comparable in both vehicles. All
compounds concentrated in intestines; intestinal concentration
remained high even after 8 h exposure. Given that no
nondissolved materials have been observed in the intestines,
the compounds likely permeated the mucosa of the gut from
the apical to the basolateral side. All compounds accumulated
in liver and lung were notable in heart and fat and detectable in
skeletal muscle and brain. Compound 11 crossed the blood−
brain barrier and was detected in brain 2 h postexposure,
although its concentration dropped to almost zero after 8 h.
The concentration of 7 in all tissues remained higher after 8 h
versus 2 h exposure, both in Kolliphor and HPβCD. The
remaining inhibitors were detected in all the tissues after 8 h,
albeit at diminished concentrations.
Dose Response: Cyclodextrin versus Kolliphor. To
validate the choice of Kolliphor as a vehicle for the in vivo oral
administration of compounds, we selected two compounds, 5
and 12, for side-by-side comparison of their efficacy using the
same batch of parasites. To design each animal experiment, we
have considered a number of different objectives, including
time, labor, resources, and ethical aspects of animal studies,
which motivated us to limit the number of experimental
animals to the minimum needed to satisfy specific questions.
The rationale behind the selection of this pair of inhibitors was
comparable bioavailability of 12 in both vehicles and relatively
low bioavailability of 5 in HPβCD compared to Kolliphor
Anti-T. cruzi Activity by po Administration. Because of
its superior performance for the majority of the tested
analogues, Kolliphor was chosen as a vehicle to assess
bioavailability and the in vivo efficacy of compounds in four-
day animal model at two different concentrations, 25 and 50
mg/kg, administered by oral gavage, bid (Table 1). All 13
compounds were tested at 25 mg/kg, and nine of them,
depending on compound availability, were also tested at 50
mg/kg. Administered at 25 mg/kg, 12 showed the best efficacy
(90.2%), closely followed by compounds 6 (89.1%) and 11
(87.4%), whereas analogues R-2 (47.4%), 2 (41.5%), 8
(47.7%), and particularly 9 (0%) were least potent. Compound
efficacy was dose-dependent, achieving >90% inhibition for six
compounds, 3 (99.2%), 5 (91.3%), 6 (97.4%), 10 (97.6%), 11
(98.6%), and 12 (98.4%), tested at 50 mg/kg, bid The efficacy
of the least potent inhibitors, R-2 and 2, also increased,
exceeding 70% at 50 mg/kg, bid. Compound 9 was the only
one in this series lacking in vivo potency. The disconnect
between the in vitro and in vivo rank order potency observed
for R-2 and 2 is likely due to reduced oral bioavailability, as
compound 2 has been characterized as one of the compounds,
with the lowest Cmax, highest clearance and shortest half-life at
50 mg/kg (Table 3).
Collectively, the three most potent inhibitors in this series, 6,
11, and 12, belong to different scaffold variants. Compound 6
and its biaryl analogues, 3 and 5, showed >90% inhibition at 50
mg/kg, all retained 2-trifluoromethyl substituent at the terminal
phenyl ring. In vivo efficacy of the biaryl analogues lacking this
substituent had been notably attenuated. The two most active
N-arylpiperazine derivatives 11 and 12, both retaining the 4-
(
Table 3). Thus, the enhancing effect of Kolliphor in vivo, if
any, was expected to be more profound for 5. Indeed, when
administered in Kolliphor, the efficacy of 5 was higher at all
concentrations tested when compared to that in HPβCD,
whereas the efficacy of 12 was comparable in both vehicles
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Figure 3. Tissue tropism of compounds. Tissue distribution of
selected inhibitors administered orally as a single 50 mg/kg dose in
2
0% HPβCD (A) or 20% Kolliphor (B). Compound concentration
Figure 4. Anti-T. cruzi efficacy of compounds in four-day mouse
model of infection. (A) Dose−response in Kolliphor (KOL) versus
HPβCD (CD) administration for compounds 5 (empty bars) and 12
detected in a tissue after 2 (blue) or 8 (orange) hours of exposure is
plotted in μM. Tabulated data are presented in Supporting
Information, Tables S1 and S2.
(
filled bars). Compound 5 in Kolliphor was more active than in
HPβCD (p < 0.05) versus compound 12, which had comparable
activity in both vehicles (P < 0.05). (B) Dose−response for Kolliphor
administration of 5, 6, 7, and 12. Benznidazole (BNZ) served as a
positive control.
fluoro substituent at the terminal phenyl ring, proved critical for
drug−target interactions by X-ray structure analysis.
Assessment of in Vivo Potency. The disconnect between
the in vitro and in vivo rank orders of potency observed in
these studies raises questions about prioritizing the CYP51
inhibitors based solely on in vitro performance without also
considering PK/PD characteristics and hydrolytic stability, in
conjunction with assessing susceptibility to oxidative metabo-
lism. Inspection of the calculated log P values predicts a lowest
log P of 2.09 for compound 9, which has no activity in the
animal model, while log P values for compounds showing
efficacy are around 5 (Table 1). A low log P value defining
relative solubility of compounds in octanol versus water is
generally predictive of poor membrane permeability. Another
possible explanation to the lack of efficacy is that the compound
R-2 and 2. EC50 ≪ K by an order of magnitude correlated
D
with attenuated efficacy of 4 and 8. Against this trend,
compound 11 (EC ≪ K by an order of magnitude) reached
50
D
one of the highest efficacy values for this series in vivo. The
rationale behind these observations may be in the high
hydrophobicity of compounds, which interferes both with
oral bioavailability and binding affinity to the target, while EC50
in cell-based assay suffers less or may even benefit to some
extent from the elevated hydrophobicity.
Inhibitor Binding Modes by X-ray Crystallography.
The X-ray structures determined in this work provide atomic
level details of drug−target interactions both for biaryl and N-
arylpiperazine derivatives. The N-arylpiperazine inhibitor, (R)-
N-(3-(1H-indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-4-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-fluorobenza-
mide, compound 12, produced the highest resolution structure
currently available for TcCYP51 (2.04 Å, PDB ID 4C0C).
Resolution for the closely related analogue, (R)-N-(3-(1H-
indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-yl)-4-(4-(3,4-
9
acetyl group may be hydrolyzed in vivo to give the piperazine
derivative, which is inactive in cell-based assay.
A ratio between EC50 and the apparent KD values
extrapolated from the titration curves in aqueous solution
(
Supporting Information, Figure S3) may provide yet another
clue. The EC50 ≪ K was a poor sign for in vivo efficacy,
D
although this criterion is not absolute. Particularly large
differences between the two parameters (about 2−3 orders of
magnitude) coincided with poor in vivo efficacy of compounds
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Figure 5. Inhibitors in the active site of TcCYP51. (A,D) Slice through the binding site shows bound inhibitors (yellow spheres) and the protein
surface colored by hydrophobicity, hydrophobic areas are in orange and hydrophilic areas are in blue. Heme is in dark-red spheres. (B) Piperazine
group separating two phenyl rings in the 12 (yellow sticks) allows smooth bending of the long substituent along the β-sheet saddle (magenta). A
fragment of the electron density map (blue mesh) contoured at 1.2 σ delineates position of 12 at 2.04 Å resolution. Protein is in ribbon, heme is in
spheres. (C,F) Residues within 5 Å from the inhibitor (yellow sticks) are highlighted in blue, heme is in gray sticks. (E) Binding mode of 1 resembles
that of 12 (B), with fewer contacts for the long substituent at the chiral carbon center. Electron density map at 2.84 Å is contoured at 0.8 σ. Images
83
84
here and otherwise were generated using CHIMERA or PYMOL
difluorophenyl)piperazin-1-yl)-2-fluorobenzamide, compound
1, was 2.43 Å (PDB ID 4UQH). The biaryl scaffold variant
Table 4. TcCYP51 Amino Acid Point Contacts within 5 Å of
the Inhibitors
1
costructure with (R)-N-(3-(1H-indol-3-yl)-1-oxo-1-(pyridin-4-
ylamino)propan-2-yl)-2′,3,5′-trifluoro-[1,1′-biphenyl]-4-carbox-
amide, compound 1, (2.84 Å, PDB ID 4BMM) is a notable
improvement compared to 3.1 Å resolution of the previously
amino acid
residues
1 (biaryl variant)
Y103, F110
12 (N-arylpiperazine variant)
invariant in
CYP51 family
Y103, F110
45
a
characterized closely related analogue R-2. The longest
substituent at the chiral center of both scaffold variants
protrudes into the β-domain (Figure 5). Being shorter than
conserved across F48, Y116, A291, T295, F48, I72 , Y116, A291, T295,
the phyla
L356, M460
L356, M460
substrate-specific I105
I105
5
6
phylum-specific
M106, P210, V213,
F290,
F55, I79, M106, P210, V213,
F290
posaconazole, both scaffolds fit entirely within the protein
interior, with the pyridinyl moiety of the inhibitor coordinated
to the heme iron and the indole ring pointed at the heme
macrocycle. Particularly surprising was the binding modes of 11
and 12, whose longest substituents extended along the
constraint channel spanning the β-domain (Figure 5A−C)
rather than hanging in the cleft between the α- and β-domain,
more variable
F214, A287, M358,
M360, V461
I45, I70, V77, F214, A287,
M358, M360, V461
a
Highlighted in bold are points of contact which do not overlap
between two inhibitors.
45
as predicted by molecular docking (here and throughout, the
nomenclature of the P450 structural elements is according to
57−59
the generally accepted schemes
).
Drug−Target Interactions and SAR. On the basis of the
X-ray structures now available for the 4-aminopyridine-based
44−46
analogues here and elsewhere,
the invariant portion of the
small-molecule skeleton binds in the most conserved area of the
CYP51 active site adjacent to the heme macrocycle (Figure 5;
Table 4). The indole ring is bound in the energetically favorable
T-shape π−π stacking mode, with the heme macrocycle tightly
enclosed by the cluster of bulky electron-rich residues Y103,
M106, F110, Y116, and F290, adopting a single well-defined
conformation (Figure 6A). This is in contrast to the previously
reported sulfonamide derivative (PDB ID 4COH), where
flipping of the indole ring was observed. The 2-fluoro
substituent of the benzamide ring, invariant in both scaffold
groups, points toward a crevice formed by the α-domain
residues Y103, I105, M106, and M460, residing within van der
Figure 6. Interactions of the terminal phenyl ring in the N-
arylpiperazine scaffold. Fluoro-substituted edges of the terminal
phenyl ring in compounds 12 (A) and 11 (B) face residues I45,
F48, F55, and I70. Inhibitors are shown in van der Waals spheres
highlighted in yellow. van der Waals radii of the amino acid residues
(blue sticks) are marked by blue dots. Heme is in stick mode.
Heteroatoms are colored by type: oxygen in red, nitrogen in blue,
fluorine in cyan, iron in ochre.
46
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Waals distances of Y103 and I105. The 2-fluoro substituent on
this ring was retained earlier in the hit-to-lead optimization for
the increased half-life of compounds in the microsome extracts.
No H-bonding interactions, direct or mediated by water
molecules, have been established between the protein and
inhibitor in any costructures reported herein. As previously
observed for the sulfonamide derivative, a single water
molecule in the active site invariantly H-bonds two amide
protons of the inhibitor.
Binding of the N-Arylpiperazine Compounds 11 and 12.
Despite the small differences in modification pattern of the
terminal phenyl ring, both N-arylpiperazine compounds had the
same binding mode. In both structures, positions of the fluorine
substituents were unambiguously defined in the electron
density, providing detailed information on drug−target
interactions. The flexibility conferred by the piperazine ring
allows 11 and 12 to adopt a smooth curvature to efficiently fill
the hydrophobic channel extending into the β-domain (Figure
that the ring in 1 is facing the opening to the exterior and that
the FG-loop possesses a large degree of flexibility to adjust to
the incoming inhibitors.
46
Most of the amino acid residues concentrated around the
invariant N-indolyl-oxopyridinyl-4-aminopropanyl part of the
skeleton are either invariable in the CYP51 protein family
(Y103 or F110) or are highly conserved across phyla (Table 4).
This suggests that the N-indolyl-oxopyridinyl-4-aminopropanyl
warhead loaded with different substituents could be utilized
against a variety of CYP51 targets. Specificity to the target is to
be defined by the structure of the variable substituent. Thus, the
terminal difluorophenyl ring of N-arylpiperazine analogues
binds in the remote cavity formed by the residues least
conserved across the CYP51 protein family compared to the
rest of the 24 exclusively hydrophobic residues constituting the
binding site. This lack of conservation suggests that N-
arylpiperazine inhibitors tailored to fit this cavity would be
likely T. cruzi specific. Compounds constituting the biaryl
scaffold may be more cross-reactive.
46
5A−C). The terminal phenyl ring critical for the in vivo efficacy
of the N-arylpiperazine analogues reached the most remote
hydrophobic cavity at the end of the channel, with the fluoro-
substituted edge facing I45, F48, F55, and I70 (Figure 6). In
both inhibitors, the 4-fluoro substituent points at I70 and I79
and is within 5 Å of I70, I72, V77, I79, and F55. The 2-fluoro
substituent of compound 12 points at I45 and is within 5 Å of
I45 and F48. Finally, the 3-fluoro substituent of compound 11
points at F55 and is within 5 Å of F48 and F55. These tight
interactions of the terminal phenyl ring carrying small fluorine
substituents may impose a binding challenge to N-arylpiper-
azine analogues featuring larger substituents, particularly at the
C-4 site.
DISCUSSION
■
The structure-aided tailoring of the N-indolyl-oxopyridinyl-4-
aminopropanyl-based scaffold described here and in our
previous publications
4
2,44−46,52
is aimed at developing drug
candidates to prevent or treat Chagas disease. Chagas disease is
an endemic tropical infection mostly affecting people in Central
and South America in areas with poor living conditions and
inadequate healthcare infrastructure. However, it is now an
emerging infection in the U.S., Spain, and Japan. In this work,
pharmacokinetic and pharmacodynamic properties of newly
developed inhibitors were monitored as part of an iterative
approach toward molecular design. This involves rounds of
analysis of structure−activity and structure−property relation-
ships, compound synthesis, testing, and re-evaluation in order
to obtain one or more classes of inhibitors that are orally
Protruding deeper into the β-domain, N-arylpiperazine
moiety induces the FG-loop conformation in TcCYP51
46
different than that observed in the sulfonamide analogue:
the loop shifts in an opposite direction, narrowing the gap
between the α- and β-domains and virtually “squeezing” the
molecules into the narrow channel. Remarkably, the twisted
shape of the N-arylpiperazine moiety in the TcCYP51 active
44−46
deliverable and potent.
An important observation made in this study is the
disconnect between the in vitro and in vivo potency of
compounds. The compounds most active in vitro, R-2 and 2,
were inferior to less potent members of the scaffold group, 6,
11, and 12, in terms of in vivo performance (Table 1). A
disconnect between the in vitro and in vivo compound ranking
is not uncommon, being also observed for different targets due
to several factors, including pharmacokinetic properties of
2
site violates a coplanar, sp -hybridized arrangement of the
nitrogen-linked aryl groups even though both nitrogen atoms of
2
the piperazine were restrained in planar sp hybridization
during structure refinement. As a result, the terminal phenyl
ring is forced into virtually orthogonal orientation with respect
to the piperazine’s nitrogen plane. Given that hybridization/
conjugation at the point of ring attachment is not beneficial to
the binding mode, a conformational penalty is likely imposed,
which may be tolerated, at least in part, in exchange for the
enthalpy gained due to the expansion of the terminal ring into
the hydrophobic cavity of β-domain formed by the five
hydrophobic residues: I70, I72, V77, I79, and F55.
6
0,61
chemicals and host metabolism.
In our specific case,
discrepancy in compound ranking may be also rooted in the
conflict between the highly hydrophobic nature of the CYP51
binding site and the attenuated bioavailability of the hydro-
phobic compounds which may best satisfy the target require-
ment. This conflict brings into relief the shortcoming of
prioritizing CYP51 inhibitors by in vitro performance alone and
highlights the need for in vivo assessment in which compound
potency and bioavailability are monitored at a very early stage
of drug development. This is not to say that in vitro screening is
not valuable. It does focus attention on a subset of compounds
that are antiparasitic versus the many that are not. The
shortcoming is in hit prioritization for in vivo proof of principle
and is not unexpected.
Binding of the Biaryl Compound 1. The net contacts for 1
in TcCYP51 are less extensive than those of 11 or 12 (Figure
5D−F). Particularly notable is loss of the multiple hydrophobic
interactions at the end of the constricted hydrophobic channel
in the β-domain due to the smaller size of the compound 1
molecule. Attenuated interactions with the target likely account
for a compromise resolution of the biaryl analogue cocrystals, as
44
reported here and previously. The terminal phenyl ring of 1 is
stabilized by stacking interactions with F48 and F214 and by
the π−electron systems of M358 and M360. The proximity of
these residues to the terminal phenyl ring of 1 may interfere
with the ring modification pattern, however, spatial constraints
are not nearly as tight as in N-arylpiperazine analogues, given
To rapidly assess the effect of drugs on the parasite load in
vivo, a four-day mouse model utilizing T. cruzi Y luc strain
expressing firefly luciferase was adopted. To impart rigor to the
test, oral bioavailability was set ab initio as a requirement. The
total duration of the assay was 8 days, including establishing
I
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acute infection in mice (3 days), dosing (4 days), and
evaluation of outcome (1 day). The parental compound
LP10, that previously demonstrated potency in a long-term
mouse model with 30 days of treatment, could not compete
with its optimized descendants in the four-day treatment model
utilized here, confirming that the bar is set high enough to
ensure success of optimization steps and rapid prioritization of
compounds entering longer-term dosing studies.
16 h (administered in Kolliphor) (Table 3). For comparison,
posaconazole and the ravuconazole prodrug E1224, used
clinically in the treatment of invasive fungal infections and
candidates for etiological treatment of Chagas disease, have
52
6
5,66
half-lives in humans measured in days.
We routinely
monitored the half-lives of all the synthesized compounds in
vitro using microsome extracts (Table 1). Over the course of
hit-to-lead optimization, a substantial increase in the half-life
44
The transgenic T. cruzi strains that have been established in
recent years accelerate drug discovery by providing several
advantages over manual counting of parasites in mouse
has been achieved compared to the parental compound LP10,
but room for improvement remains. In future efforts, analysis of
metabolites will be undertaken to identify “weak” points that
can be addressed by further medicinal chemistry optimization
to alleviate metabolic liabilities that are uncovered.
6
2,63
blood.
However, our results also show that transgenic T.
cruzi parasites expressing firefly luciferase are less virulent than
the parental strain because mice survive infection with a
relatively high inoculum that would be lethal with the parental
strain. Similar observations have been made with transgenic T.
cruzi parasites with a different background, which develop lower
The persistent cross-reactivity of compounds with CYP2C9
is noteworthy given that another human isoform, 3A4, is a
major CYP off-target for the azole antifungal drugs,
ketoconazole and posaconazole. The IC50 of ketoconazole
and posaconazole reported against human 3A4 vary in different
64
parasitemia when compared to wild type. This variability
renders T. cruzi Y luc strain unsuitable either for long-term
treatment schemes or evaluation of parasitological cure.
Nevertheless, the rapid and sensitive detection of the
luciferase-expressing parasites makes it a valuable tool for
assessing compound bioavailability and efficacy in the early
stages of drug discovery.
35
67
publications as 40 and 350 nM, or 8 and 120 nM. 3A4 is
highly promiscuous in substrate specificity and oxidizes over
68
half of all administered drugs. Unlike 3A4, 2C9 exhibits
selectivity for the oxidation of relatively small, lipophilic anionic
compounds such as the nonsteroidal anti-inflammatory drugs
flurbiprofen, ibuprofen, naproxen, and diclofenac as well as the
hypolipidemic agent gemfibrozil. Arginine 108 is a point of
The nonionic surfactant Kolliphor, which has low reported in
5
5
69
vivo toxicity, was utilized as a vehicle for oral administration
to assess the solubility, efficacy, and systemic and tissue
exposure of the compounds. Use of Kolliphor formulations was
proven superior to HPβCD for the tested set of the compounds
as far as drug solubility and exposure are concerned (Table 3).
Efficacy in the four-day animal model (Figure 4A) was
consistent with exposure: compound 5 formulated in Kolliphor
was more active compared to the HPβCD formulation for
which large difference in drug exposure was also observed
between the two vehicles. Compound 12 had comparable
exposure and efficacy in both formulation vehicles. No signs of
toxicity or side effects have been observed in the four-day
studies.
specific contact stabilizing the charge of these anionic drugs.
On the basis of the X-ray structure analysis, 2C9 has a large and
hydrophobic binding site capable of simultaneously binding
69,70
multiple ligands.
Also, structural conformational changes
upon interactions with the ligand allow 2C9 to bind molecules
6
9,70
of various sizes and chemical structures.
Given that the
longest substituent at the chiral carbon center was an
optimization target in the N-indolyl-oxopyridinyl-4-amino-
44−46
propanyl-based SAR series, here and previously reported,
persistent cross-reactivity with 2C9 may be associated with the
invariable portion of the skeleton rather than variable
substituent. Another pyridine-based class of CYP51 inhibitors,
fenarimol analogues EPL-BS967 (also known as UDD) and
3
7−39
Another important observation from these studies is that
small modifications in molecular structure may have dramatic
effects on both in vitro and in vivo parameters of the inhibitors.
Thus, modifications to the terminal phenyl ring resulted in a
wide range of potencies of the biaryl scaffold (Table 1).
Similarly, PK parameters of the N-arylpiperazine analogues
differed significantly from one another (Table 3). Given the
multiple factors affecting the efficacy of drugs, the two most
efficacious and competitive compounds, 6 and 12, represented
different scaffold groups. Improved microsome stability in vitro
demonstrated by biaryl compound 6 contrasted with the
attenuated microsome stability of N-arylpiperazine member 12.
Compared to 12, compound 6 had higher Cmax and AUC,
which was consistent with its low clearance. On the other hand,
compound 12 had longer terminal half-life in plasma and a
distinct, tight drug−target fit which distinguished it from the
CYP51 inhibitors of the biaryl series. However, the near-perfect
space-filling of 12 binding at the active site suggests a
EPL-BS1246 (also known as UDO),
have IC s of 8−9
5
0
67
μM and ≥20 μM, toward 2C9 and 3A4, respectively.
Although both N-indolyl-oxopyridinyl-4-aminopropanyl-
based and fenarimol analogues utilize a six-membered pyridine
heterocycle to coordinate the heme iron (a role consistent with
that of the five-membered heterocycle in azole inhibitors), the
two pyridine-based scaffolds have significant differences
defining their interactions with the target (Figure 7). First,
the 4-pyridyl Fe-coordinating moiety of the N-indolyl-
oxopyridinyl-4-aminopropanyl-based analogues contrasts to
the 3-pyridyl moiety of fenarimol analogues. Second, the
structure branching point (highlighted in green in Figure 7) in
the fenarimol analogues is positioned three bond-lengths closer
to the Fe-coordinating nitrogen atom compared to that in the
N-indolyl-oxopyridinyl-4-aminopropanyl-based analogues. Both
factors synergize to impose spatial constraints on the
interactions of the fenarimol analogues with the heme
macrocycle. Structurally, it translates to the energetically
more favorable T-shape π−π stacking interactions between
the indole ring and heme macrocycle in the N-indolyl-
oxopyridinyl-4-aminopropanyl-based inhibitors (Figure 7A),
as opposed to spatial hindrance caused by proximity of virtually
coplanar aromatic systems of heme and fenarimol analogues
(Figure 7B). This steric hindrance rather than the pyridine
group electronic effects likely explains a weakened Fe−N
2
conformational penalty for violating the coplanar, sp -
hybridized arrangement of the nitrogen-linked aryl groups in
the piperazine moiety. Finding chemical alternative(s) to the
piperazine linker may alleviate the restraint and further enhance
binding to TcCYP51.
The half-lives of N-indolyl-oxopyridinyl-4-aminopropanyl-
based analogues tested in these rodent studies ranged from 3 to
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methodologies, and tools necessary to successfully monitor
each aspect of lead improvement. Valuable additions to the
toolbox include an application of the T. cruzi Y strain
expressing firefly luciferase for quick assessment of bioavail-
ability and potency and inclusion of high-resolution crystal
structures as plug-ins for analysis of the pharmacodynamic
aspects of lead optimization. The new class of molecules has
considerable promise for the development of anti-T. cruzi
therapeutics and represents the current trend in the field of
drug discovery for Chagas disease toward lead diversification
and optimization specifically directed against the T. cruzi
molecular target.
EXPERIMENTAL SECTION
Chemistry. General Procedure for the Synthesis of Inhibitors 1,
, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. To a solution of the appropriate
■
2
benzoic acid (ca.1.2 equiv), PyBOP (ca. 1.4 equiv), and HOBt (ca. 10
mol %) in dry CH Cl (5 mL) was slowly added triethylamine (ca. 4
2
2
equiv) at ambient temperature. The reaction mixture was stirred for 15
45
min until it became homogeneous. D-Tryptophan derivative 30 was
added, and the reaction mixture was stirred at room temperature for 1
h. After confirming that the reaction was complete by using TLC
analysis, the solvent was removed under reduced pressure. Ethyl
acetate (10 mL) was added to the crude product mixture, and this
solution was washed then with saturated aqueous NaHCO (2 mL ×
3
2
) and brine (2 mL × 2). The organic layer was concentrated in vacuo,
and the crude product was directly subjected to flash chromatography
purification to provide the titled products in ca. 70% yield.
(
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-2′,3,5′-trifluoro-[1,1′-biphenyl]-4-carboxamide TFA (1). The
general procedure was followed using carboxylic acid 13 as the
acylating agent. The crude product was further purified by HPLC to
Figure 7. Pyridine-based CYP51 inhibitors. (A) N-Indolyl-oxopyr-
idinyl-4-aminopropanyl-based analogues. (B) Fenarimol analogues.
Drug−heme van der Waals interactions are shown as resolved in the
X-ray structures of the corresponding drug−target complexes (PDB
ID codes are in parentheses). Heme is in gray van der Waals spheres;
inhibitors colored by atom types with carbon highlighted in yellow are
labeled by the small-molecule codes. The branching points in chemical
structures are highlighted in green.
26
1
afford 1 as a white solid (69%): [α]D = −70.0 (c = 1.0, MeOH). H
NMR (400 MHz, DMSO-d ) δ 11.55 (s, 1H), 10.95 (d, J = 2.5 Hz,
6
1
H), 8.83 (dd, J = 6.9, 2.7 Hz, 1H), 8.76−8.58 (m, 2H), 8.15−7.92
(
9
1
m, 2H), 7.67 (q, J = 7.9 Hz, 2H), 7.59−7.48 (m, 3H), 7.43 (td, J =
.5, 4.6 Hz, 1H), 7.38−7.26 (m, 3H), 7.06 (ddd, J = 8.1, 7.0, 1.2 Hz,
13
H), 7.00−6.92 (m, 1H), 4.98−4.85 (m, 1H), 3.42−3.17 (m, 2H).
C
NMR (101 MHz, DMSO-d ) δ 172.77, 163.59, 160.46, 159.53, 157.98,
6
1
1
57.15, 156.44, 154.03, 151.61, 143.78, 138.38, 138.29, 136.09, 130.50,
30.47, 127.08, 124.96, 124.92, 124.88, 124.14, 122.71, 122.57, 121.04,
coordination bond in the fenarimol analogue−TcCYP51
complexes, which is >0.2 Å longer than that in the N-indolyl-
oxopyridinyl-4-aminopropanyl-based complexes and >0.3 Å
118.38, 118.33, 118.14, 118.05, 117.89, 117.80, 117.16, 117.12, 116.89,
116.71, 116.68, 116.45, 114.45, 111.41, 109.00, 55.69, 26.99. MS (ESI)
m/z 515.2 [M + H] . HRMS (ESI) m/z for C29
calcd 515.1695, found 515.1696.
67
+
[M + H]+
longer than in azoles. Attenuated metal-binding is believed to
improve selectivity and safety profile of the fenarimol
analogues, compared to that of azole antifungal inhibitors, by
increasing contributions of specific drug−protein interactions in
binding affinity at the expense of nonspecific drug−metal
interactions. A similar approach has been used to achieve highly
selective CYP17A1 inhibitors, a potential treatment for prostate
H
22
N
4
O
2
F
3
(
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-3,3′,4′-trifluoro-[1,1′-biphenyl]-4-carboxamide (2). The general
procedure was followed using 14 as the acylating agent to provide 2 as
26
1
a light-yellow solid (80%): [α]D = −69.3 (c = 1.0, MeOH). H NMR
400 MHz, DMSO-d ) δ 10.90 (d, J = 2.5 Hz, 1H), 10.68 (s, 1H), 8.56
(
6
(dd, J = 7.4, 3.7 Hz, 1H), 8.51−8.38 (m, 2H), 7.93 (ddd, J = 12.1, 7.7,
2.3 Hz, 1H), 7.77−7.50 (m, 8H), 7.33 (d, J = 8.1 Hz, 1H), 7.25 (d, J =
2.3 Hz, 1H), 7.13−7.02 (m, 1H), 6.97 (t, J = 7.4 Hz, 1H), 4.91 (td, J =
71
cancer, via utilizing less avid metal-binding groups. Accord-
ingly, in future studies we will explore analogues of our N-
indolyl-oxopyridinyl-4-aminopropanyl-based inhibitors with
less nucleophilic metal-coordinating heterocycles in an attempt
to increase CYP51 selectivity of this class of inhibitors.
13
8
.2, 5.6 Hz, 1H), 3.43−3.16 (m, 2H). C NMR (101 MHz, DMSO-
d ) δ 171.52, 163.22, 161.08, 158.60, 151.09, 151.05, 150.96, 150.92,
6
1
1
1
50.32, 148.64, 148.58, 148.52, 148.46, 145.53, 142.34, 142.24, 136.07,
35.30, 130.98, 130.95, 127.19, 123.94, 123.89, 123.86, 122.56, 122.53,
22.09, 121.95, 121.00, 118.50, 118.28, 118.18, 118.01, 116.30, 116.12,
CONCLUSIONS
■
114.46, 114.21, 113.45, 111.35, 109.35, 55.14, 27.43. MS (ESI) m/z
+
+
The 4-aminopyridyl-based molecular scaffold possesses features
key for translating lead compounds into clinical drug
candidates: potency against the therapeutic target, large
therapeutic window, oral bioavailability, and high tissue
tropism, while terminal half-life and cross-reactivity with
human CYPs need further optimization. Only concomitant
enhancement of each of these features may allow improvements
sufficient to ensure parasitological cure in mice and humans.
Over the course of this work, we have assembled drug leads,
515.2 [M + H] . HRMS (ESI) m/z for C H N O F [M + H] calcd
515.1695, found 515.1695.
29 22 4 2 3
(R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-3-fluoro-3′-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxamide hy-
drochloride (3). The general procedure was followed using 15 as
the acylating agent to provide 3 as a light-yellow solid (73%), which
was further purified by HPLC. The HCl salt of 3 was obtained by
adding 2 N HCl (ca. 1 mL) to 3 in aqueous solution during
evaporation: [α]D26 = −73.1 (c = 1.0, MeOH). H NMR (400 MHz,
1
DMSO-d ) δ 15.44 (s, 1H), 12.24 (s, 1H), 11.00 (d, J = 2.4 Hz, 1H),
6
K
dx.doi.org/10.1021/jm500448u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
8
7
.84−8.65 (m, 3H), 8.27−8.16 (m, 2H), 8.13−8.03 (m, 2H), 7.87−
.65 (m, 6H), 7.39−7.29 (m, 2H), 7.10−7.00 (m, 1H), 6.95 (t, J = 7.4
1H), 7.76−7.67 (m, 3H), 7.64 (dd, J = 8.1, 1.7 Hz, 1H), 7.53 (t, J = 9.0
Hz, 1H), 7.47 (t, J = 100.0 Hz, 1H), 7.39−7.29 (m, 2H), 7.04 (ddd, J
= 8.1, 6.9, 1.1 Hz, 1H), 6.99−6.88 (m, 1H), 4.98 (ddd, J = 8.8, 6.8, 5.4
13
Hz, 1H), 4.98 (ddd, J = 8.7, 6.8, 5.4 Hz, 1H), 3.50−3.22 (m, 2H).
NMR (101 MHz, DMSO-d ) δ 173.04, 163.45, 161.16, 158.68, 152.96,
C
13
Hz, 1H), 3.51−3.23 (m, 2H). C NMR (101 MHz, DMSO-d ) δ
6
6
1
1
1
1
+
43.09, 143.00, 142.16, 138.82, 136.08, 131.12, 131.06, 131.03, 130.23,
30.10, 129.78, 127.14, 125.46, 125.22, 124.23, 123.57, 123.54, 123.50,
23.46, 122.91, 122.88, 122.75, 122.14, 122.00, 120.99, 118.55, 118.32,
14.82, 114.60, 111.38, 108.99, 55.94, 26.97. MS (ESI) m/z 547.2 [M
173.02, 163.40, 161.12, 158.72, 158.64, 156.25, 152.96, 142.32, 142.24,
142.12, 136.07, 135.54, 130.99, 130.96, 129.10, 127.79, 127.71, 127.14,
124.22, 122.66, 122.63, 121.82, 121.69, 120.98, 120.42, 120.24, 118.56,
118.31, 117.57, 117.36, 114.59, 114.33, 111.37, 108.98, 55.93, 26.97.
+
+
+
H] . HRMS (ESI) m/z for C H N O F [M + H] calcd
MS (ESI) m/z 531.1 [M + H] . HRMS (ESI) m/z for
30
23
4
2 4
+
547.1757, found 547.1756.
C H N O F Cl [M + H] calcd 531.1399, found 531.1391.
29
22
4
2 2
(
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
(R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-3,3′-difluoro-4′-((4-fluorobenzyl)oxy)-[1,1′-biphenyl]-4-carboxa-
yl)-2-fluoro-4-(4-(3-fluorophenyl)piperazin-1-yl)benzamide (8). The
general procedure was followed using 26b as the acylating agent to
mide (4). The general procedure was followed using 22b as the
26
acylating agent to provide 4 as a light-yellow solid (85%): [α]D
−
=
26
provide 8 as a light-yellow solid (84%): [α]D = −61.2 (c = 1.0,
1
54.6 (c = 1.0, MeOH). H NMR (400 MHz, DMSO-d ) δ 10.90 (d, J
1
6
MeOH). H NMR (400 MHz, DMSO-d ) δ 10.89 (d, J = 2.4 Hz, 1H),
6
=
2.5 Hz, 1H), 10.67 (s, 1H), 8.55−8.39 (m, 3H), 7.73 (dd, J = 12.8,
1
(
8
(
0.65 (s, 1H), 8.49−8.38 (m, 2H), 7.81 (t, J = 7.5 Hz, 1H), 7.67−7.53
m, 4H), 7.33 (d, J = 8.1 Hz, 1H), 7.28−7.18 (m, 2H), 7.05 (ddd, J =
.1, 6.9, 1.2 Hz, 1H), 6.99−6.91 (m, 1H), 6.90−6.73 (m, 4H), 6.57
td, J = 8.4, 1.8 Hz, 1H), 4.95−4.83 (m, 1H), 3.43 (dd, J = 6.9, 3.6 Hz,
2
.3 Hz, 1H), 7.70−7.56 (m, 7H), 7.56−7.51 (m, 2H), 7.35 (t, J = 8.6
Hz, 2H), 7.29−7.19 (m, 3H), 7.06 (ddd, J = 8.0, 6.9, 1.1 Hz, 1H),
7
3
1
1
1
1
1
.01−6.86 (m, 1H), 5.23 (s, 2H), 4.91 (td, J = 8.1, 5.6 Hz, 1H), 3.34−
.19 (m, 2H). 13C NMR (101 MHz, DMSO-d ) δ 171.53, 163.25,
13
6
4H), 3.39−3.19 (m, 6H). C NMR (101 MHz, DMSO-d ) δ 171.69,
6
63.13, 161.21, 160.71, 158.74, 153.26, 150.84, 150.39, 146.52, 146.42,
45.47, 143.12, 143.04, 136.08, 132.65, 132.62, 130.95, 130.92, 130.86,
30.18, 130.10, 127.19, 123.94, 123.18, 123.15, 122.01, 121.98, 121.22,
21.08, 121.00, 118.50, 118.28, 115.76, 115.48, 115.27, 114.68, 114.49,
13.79, 113.55, 113.45, 111.36, 109.34, 69.55, 55.12, 27.45. MS (ESI)
1
1
1
1
4
64.48, 162.95, 162.92, 162.70, 162.09, 160.26, 154.15, 154.03, 152.48,
52.38, 150.37, 145.49, 136.10, 131.74, 131.70, 130.44, 130.34, 127.21,
23.92, 121.01, 118.45, 118.29, 113.42, 111.36, 111.00, 110.18, 110.05,
09.84, 109.22, 105.08, 104.87, 102.09, 101.84, 100.85, 100.57, 54.91,
+
7.21, 46.42, 27.57. MS (ESI) m/z 581.5 [M + H] . HRMS (ESI) m/z
+
+
m/z 621.3 [M + H] . HRMS (ESI) m/z for C H N O F [M + H]
+
36
28
4
3
3
for C H N O F [M + H] calcd 581.2477, found 581.2480.
33
31
6
2 2
calcd 621.2114, found 621.2115.
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-2′,3-difluoro-5′-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxamide
5). The general procedure was followed using 16 as the acylating
(
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
(
yl)-4-(4-acetylpiperazin-1-yl)-2-fluorobenzamide Hydrochloride (9).
The general procedure was followed using 24b as the acylating agent
to provide 9, which was further purified by HPLC to afford the
product as a white solid. The HCl salt of 9 was obtained by adding 2 N
HCl (ca. 1 mL) to 9 in aqueous solution during evaporation (67%):
(
27
agent to provide 5 as a light-yellow solid (85%): [α]D = −57.6 (c =
1
0
1
7
7
7
6
.54, MeOH). H NMR (400 MHz, DMSO-d ) δ 10.90 (d, J = 2.5 Hz,
6
H), 10.68 (s, 1H), 8.67 (dd, J = 7.4, 3.2 Hz, 1H), 8.51−8.40 (m, 2H),
.98 (dd, J = 7.1, 2.4 Hz, 1H), 7.89 (ddd, J = 8.5, 4.4, 2.4 Hz, 1H),
.76−7.66 (m, 2H), 7.66−7.57 (m, 4H), 7.54 (dt, J = 8.0, 1.7 Hz, 1H),
.34 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.07 (ddd, J = 8.1,
.9, 1.2 Hz, 1H), 7.02−6.92 (m, 1H), 4.99−4.83 (m, 1H), 3.39−3.16
26
1
[
α]D = −57.3 (c = 1.0, MeOH). H NMR (400 MHz, DMSO-d ) δ
6
1
2
7
5.42 (s, 1H), 12.25 (s, 1H), 10.99 (d, J = 2.5 Hz, 1H), 8.80−8.65 (m,
H), 8.26−8.14 (m, 2H), 7.95 (dd, J = 8.4, 5.6 Hz, 1H), 7.67 (d, J =
.9 Hz, 1H), 7.60 (t, J = 9.0 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.29 (d,
m, 2H). 1 C NMR (101 MHz, DMSO-d ) δ 171.48, 163.26, 162.31,
3
J = 2.3 Hz, 1H), 7.04 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H), 6.95−6.88 (m,
(
6
1
6
H), 6.84−6.72 (m, 2H), 4.92 (dt, J = 7.8, 5.8 Hz, 1H), 3.54 (dd, J =
1
1
1
1
1
60.47, 159.78, 157.98, 150.38, 145.48, 137.75, 137.66, 136.07, 130.59,
30.56, 128.21, 128.18, 128.14, 127.77, 127.38, 127.25, 127.19, 126.19,
26.16, 125.87, 125.83, 125.14, 125.11, 125.06, 123.94, 123.08, 122.93,
22.35, 120.99, 118.50, 118.28, 117.78, 117.54, 116.93, 116.70, 116.67,
13
.7, 3.9 Hz, 4H), 3.45−3.23 (m, 6H), 2.03 (s, 3H). C NMR (101
MHz, DMSO-d ) δ 173.26, 168.43, 163.16, 163.13, 162.73, 160.28,
6
1
54.09, 153.98, 153.04, 142.09, 136.11, 131.70, 131.66, 127.12, 124.19,
+
121.02, 118.50, 118.33, 114.55, 111.40, 109.96, 109.84, 109.80, 108.88,
13.45, 111.35, 109.36, 55.16, 27.44. MS (ESI) m/z 565.2 [M + H] .
+
100.86, 100.57, 55.71, 46.77, 46.45, 44.91, 26.99, 21.21. MS (ESI) m/z
HRMS (ESI) m/z for C H N O F [M + H] calcd 565.1663, found
30
22
4
2
5
+
+
5
29.3 [M + H] . HRMS (ESI) m/z for C H N O F [M + H] calcd
29 30 6 3
5
65.1658.
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-3,4′-difluoro-3′-(trifluoromethyl)-[1,1′-biphenyl]-4-carboxamide
6). The general procedure was followed using 17 as the acylating
5
29.2363, found 529.2365.
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
(
(
yl)-4-(4-(3-chlorophenyl)piperazin-1-yl)-2-fluorobenzamide (10).
The general procedure was followed using 27b as the acylating
agent to provide 10 as a light-yellow solid (61%): [α] = −65.7 (c =
(
27
agent to provide 6 as a light-yellow solid (68%): [α]D = −69.6 (c =
2
D
7
1
0
1
8
7
.56, MeOH). H NMR (400 MHz, DMSO-d ) δ 10.90 (d, J = 2.5 Hz,
6
1
0
1
7
.72, MeOH). H NMR (400 MHz, DMSO-d ) δ 10.90 (d, J = 2.5 Hz,
H), 10.70 (s, 1H), 8.53−8.39 (m, 2H), 7.82 (t, J = 7.5 Hz, 1H),
.68−7.58 (m, 4H), 7.33 (d, J = 8.1 Hz, 1H), 7.27−7.19 (m, 2H), 7.05
H), 10.73 (s, 1H), 8.59 (dd, J = 7.3, 3.7 Hz, 1H), 8.50−8.42 (m, 2H),
.22−8.06 (m, 2H), 7.82−7.75 (m, 1H), 7.74−7.66 (m, 4H), 7.66−
.60 (m, 3H), 7.34 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.06
6
(
ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 7.00 (t, J = 2.2 Hz, 1H), 6.97−6.90 (m,
(
ddd, J = 8.0, 6.9, 1.2 Hz, 1H), 7.01−6.93 (m, 1H), 4.92 (td, J = 7.9,
.5 Hz, 1H), 3.39−3.17 (m, 2H). C NMR (101 MHz, DMSO-d ) δ
13
2H), 6.88−6.76 (m, 3H), 4.98−4.82 (m, 1H), 3.43 (dd, J = 6.8, 3.7
5
1
1
1
1
2
6
13
Hz, 4H), 3.37−3.19 (m, 6H). C NMR (101 MHz, DMSO-d ) δ
71.57, 163.23, 161.09, 160.30, 158.60, 150.05, 145.79, 142.06, 141.98,
36.09, 134.87, 133.88, 133.79, 131.04, 131.01, 127.20, 125.82, 125.77,
23.95, 123.89, 122.87, 122.84, 122.29, 122.15, 121.17, 121.00, 118.49,
18.29, 118.08, 117.87, 114.79, 114.55, 113.50, 111.36, 109.34, 55.19,
6
1
1
1
1
71.77, 162.96, 162.94, 162.70, 160.26, 154.14, 154.03, 151.91, 150.01,
45.82, 136.10, 133.86, 131.74, 131.70, 130.48, 127.20, 123.93, 121.01,
18.45, 118.36, 118.29, 114.74, 113.84, 113.47, 111.37, 110.17, 110.04,
+
+
09.83, 109.20, 100.85, 100.57, 54.95, 47.21, 46.42, 27.54. MS (ESI)
7.43. MS (ESI) m/z 565.2 [M + H] . MS (ESI) m/z 565.2 [M + H] .
+
+
m/z 597.2 [M + H] . HRMS (ESI) m/z for C H N O FCl [M +
H] calcd 597.2181, found 597.2188.
HRMS (ESI) m/z for C H N O F [M + H] calcd 565.1663, found
33 31
6
2
30
22
4
2 5
+
565.1664.
(
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
(
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-4-(4-(3,4-difluorophenyl)piperazin-1-yl)-2-fluorobenzamide (11).
The general procedure was followed using 28b as the acylating agent
yl)-3′-chloro-3,4′-difluoro-[1,1′-biphenyl]-4-carboxamide hydro-
chloride (7). The general procedure was followed using 18 as the
acylating agent to provide 7 as a light-yellow solid, which was further
purified by HPLC. The HCl salt of 7 (58%) was obtained by adding 2
27
D
to provide 11 as a light-yellow solid (64%): [α]
MeOH). H NMR (400 MHz, DMSO-d
= −69.5 (c = 0.46,
) δ 10.89 (d, J = 2.5 Hz, 1H),
6
1
27
N HCl (ca. 1 mL) to 7 in aqueous solution during evaporation: [α]D
10.70 (s, 1H), 8.52−8.36 (m, 2H), 7.82 (t, J = 7.5 Hz, 1H), 7.69−7.55
(m, 4H), 7.33 (d, J = 8.1 Hz, 1H), 7.27 (dt, J = 10.7, 9.4 Hz, 1H), 7.21
(d, J = 2.4 Hz, 1H), 7.11−7.01 (m, 2H), 6.93 (t, J = 7.4 Hz, 1H),
6.89−6.75 (m, 3H), 4.99−4.80 (m, 1H), 3.43 (dd, J = 6.8, 3.7 Hz,
1
=
−54.9 (c = 0.57, MeOH). H NMR (400 MHz, DMSO-d ) δ 12.29
6
(
s, 1H), 11.00 (d, J = 2.5 Hz, 1H), 8.79−8.67 (m, 3H), 8.27−8.17 (m,
H), 8.03 (dd, J = 7.1, 2.4 Hz, 1H), 7.79 (ddd, J = 8.7, 4.7, 2.4 Hz,
2
L
dx.doi.org/10.1021/jm500448u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
3
4
1
1
1
1
4
H), 3.37−3.16 (m, 6H). C NMR (101 MHz, DMSO-d ) δ 171.76,
C2C12 myoblasts (500 cells/well) and then were infected with CA-I/
72 trypomastigotes (2500 parasites/well) in 50 μL of culture medium
(DMEM H-21)/well. Culture plates were incubated at 37 °C with 5%
6
62.96, 162.93, 162.69, 160.25, 154.15, 154.04, 151.01, 150.88, 150.01,
48.60, 148.47, 148.24, 148.16, 145.82, 141.84, 136.10, 131.73, 131.68,
27.20, 123.93, 121.01, 118.45, 118.28, 117.40, 117.23, 113.47, 111.37,
10.23, 110.11, 109.91, 109.20, 104.77, 104.56, 100.94, 100.66, 54.94,
7.89, 46.48, 27.54. MS (ESI) m/z 599.2 [M + H] . HRMS (ESI) m/z
for C H N O F [M + H] calcd 599.2382, found 599.2374.
R)-N-(3-(1H-Indol-3-yl)-1-oxo-1-(pyridin-4-ylamino)propan-2-
yl)-4-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-fluorobenzamide (12).
The general procedure was followed using 29b as the acylating agent
to provide 12 as a light-yellow solid (74%): [α]D = −52.8 (c = 0.61,
MeOH). H NMR (400 MHz, DMSO-d ) δ 10.89 (d, J = 2.5 Hz, 1H),
CO . Twenty-four hours postinfection, culture medium was removed
2
and test compounds were added in fresh medium. For this, an
intermediate 384-well plate was prepared by serial dilution for all the
compounds in 100% DMSO. Then, 50 nL of each sample were diluted
in 50 μL of media (DMEM H-21) to final concentrations of 10 μM, 2
μM, 400 nM, 80 nM, 16 nM, 3 nM, 128 pM, 25 pM, and 5 pM and
added to the experimental plate followed by incubation at 37 °C with
+
+
33
30
6
2 3
(
27
5% CO for 72 h. Wells containing noninfected cells were used as a
2
1
6
positive control (100% cell survival), while T. cruzi-infected but
untreated cells (0% cell survival) were used as a negative control.
Cells were then fixed for 2 h with 4% paraformaldehyde and rinsed
1
(
(
0.64 (s, 1H), 8.50−8.38 (m, 2H), 7.82 (t, J = 7.5 Hz, 1H), 7.69−7.56
m, 4H), 7.33 (dt, J = 8.1, 0.9 Hz, 1H), 7.28−7.18 (m, 2H), 7.16−6.97
m, 3H), 6.94 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.89−6.77 (m, 2H),
with a solution of 150 mM NaCl, 100 mM NH Cl, 0.1% Triton X-100,
4
4
2
1
1
1
1
1
1
.96−4.81 (m, 1H), 3.44 (dd, J = 6.7, 3.5 Hz, 4H), 3.34−3.19 (m,
and 0.1% NaN After that, they were treated for 4 h with 0.2 μg/mL of
3.
13
H), 3.16−3.01 (m, 4H). C NMR (101 MHz, DMSO-d ) δ 171.69,
the DNA fluorescent dye, DAPI (4,6-diamidino-2-phenylindole),
diluted in the same solution. Plates were kept at ambient temperature
until image acquisition was performed. Images were acquired by an IN
Cell Analyzer 2000 (GE Healthcare), and the procedure and analyses
6
62.95, 162.92, 162.68, 160.24, 158.48, 156.15, 156.09, 156.03, 155.96,
54.30, 154.19, 153.69, 153.56, 150.36, 145.50, 136.48, 136.44, 136.39,
36.35, 136.10, 131.72, 131.67, 127.21, 123.92, 121.01, 120.31, 120.27,
20.22, 120.18, 118.46, 118.29, 113.43, 111.37, 111.21, 111.17, 111.00,
10.96, 110.31, 110.18, 109.92, 109.22, 104.95, 104.69, 104.44, 100.95,
5
1,73
were performed according to previously described.
Cytotoxicity Assay. Cytotoxicity of compounds was assessed
+
00.67, 54.91, 50.06, 46.92, 27.57. MS (ESI) m/z 599.2 [M + H] .
HRMS (ESI) m/z for C H N O F [M + H] calcd 599.2382, found
against two different cell types, cardiomyocytes and hepatocytes,
+
75,76
33
30
6
2 3
isolated from 18-day-old mouse embryos as previously described.
5
99.2380.
Briefly, to obtain cardiomyocytes, cardiac embryo fragments were
dissociated in PBS, supplemented with 0.025% trypsin and 0.01%
collagenase (Worthington Co., Lakewood, NJ), and plated in a density
Compounds were stored at −20 °C as 10 mM stock solutions in
DMSO and freshly diluted immediately prior to use.
Hepatic Microsomal Stability. Hepatic microsomal stability was
addressed as described previously.
CYP Inhibition. Cytochrome P450 inhibition was evaluated in
human liver microsomes using four selective marker substrates as
5
of 5 × 10 cells/well into a white opaque flat bottom μclear 96-well
44
plate (Greiner Bio-One) coated with 0.01% gelatin. Cultures were
maintained in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% FBS, 2.5 mM CaCl , 1 mM L-glutamine,
2
44
described previously.
and 2% chicken embryo extract. Hepatocytes were isolated through
incubation of embryo liver fragments with 0.05% solution of
collagenase type III in Hank’s Balanced Salt Solution (HBSS): 0.14
M NaCl, 5.37 M KCl, 0.84 mM MgSO , 0.44 mM KH PO , 1,50 g/L
Binding Affinity by UV−vis Spectroscopy. Binding affinity of
compounds was approximated from the spectrophotometry titration
curves generated as previously described.
T. cruzi Maintenance. T. cruzi, Y luc strain, transformed with
46
4
2
4
glucose, 0,0005 g/L insulin, 0.02 M HEPES, pH 7.4. The liver cells
5
episomal firefly luciferase gene, was developed as described else-
were dispersed by pipetting and seeded in a density of 1.5 × 10 cells/
47
where. All media components used in this work were purchased
from Sigma unless indicated otherwise. Cultured trypomastigotes were
obtained by weekly infection of C2C12 myoblasts, with trypomasti-
gotes being released in the supernatant 4−7 days post infection,
collected by centrifugation for 15 min at 3300 rpm. Without selective
antibiotic pressure, the luciferase expression in the parasite was
detectable for about as long as seven passages in mammalian culture.
To maintain high titer of luciferase marker in parasite population, the
pressure of G418 antibiotic was applied to epimastigote form. For that,
well into a white opaque flat bottom μclear 96-well plate (Greiner Bio-
One) coated with 0.01% gelatin in hepatocyte adhesion medium (70%
MEM/30% 199 medium, supplemented with 10% FBS, 5 mM CaCl2).
After 6 h, the cultures were washed in HBSS and the media was
changed to a mixture of DMEM/199 at 70/30% ratio supplemented
with 10% FBS, 2 mM L-glutamin, 10 μg/mL insulin, 1 ng/mL
6
glucagon, 50 ng/mL EGF, 3.5 × 10 U hydrocortisone, 6.5 ng/mL
somatotropin, and 0.5 μg/mL linoleic acid. All cultures were kept at 37
°C in an atmosphere of 5% CO . For the cytotoxicity assay, the
2
72
the epimastigotes were cultivated in LIT medium, supplemented
with 10% fetal bovine serum (FBS) and 200 μg/mL of G418, at 28 °C.
Once a month, myoblast cultures were infected with 2−4 week old
epimastigotes enriched with the metacyclic trypomastigotes. Forty-
eight hours postinfection, the epimastigotes were removed from the
medium by successive washing of cultures with phosphate buffer saline
compounds were serially diluted freshly from 10 mM DMSO stock
solutions with appropriate medium and added to primary culture cells
at final concentrations of 90, 30, 10, 3.3, 1.1, 0.37, 0.12, and 0.04 μM.
After 72 h incubation, the viability of the cells was assessed using the
ATP-detection luminescence-based kit, Cell Titer Glo (Promega). The
EC50 values were determined based in the sigmoidal dose−response
curves of the cell growth inhibition. Selectivity index (SI) for the
compounds was calculated as a ratio between the mammalian cell and
T. cruzi amastigote viability expressed as corresponding EC50 values.
Four-Day Dosing Mouse Model of T. cruzi Infection. The
four-day dosing mouse model of T. cruzi infection was used to assess
in vivo potency of increasingly optimized inhibitors. Eight-week-old
female Swiss Webster albino mice (average weight 20 g) were obtained
from Simonsen Laboratories (Gilroy, CA). All animal procedures were
approved and carried out in accordance with the guidelines established
by the Institutional Animal Care and Use Committee from UCSF
(approval no. AN087605-01). Mice were housed at a maximum of five
per cage and kept in a specific-pathogen free (SPF) room at 20 to 24
°C under a 12 h light/12 h dark cycle and provided with sterilized
water and chow ad libitum. To infect the mice, trypomastigotes of T.
cruzi Y luc strain were harvested from culture supernatant and injected
(
PBS). Seven days postinfection, trypomastigote population enriched
with the transgenic parasites expressing luciferase was collected from
the medium.
Dose−Response in T. cruzi Cell-Based Assay. To estimate
compound’s anti-T. cruzi potency, the EC50 values of compounds were
7
3
determined in the cell-based assay adapted from Engel and coauthors
5
1
and modified as previously described. Briefly, mouse C2C12
myoblasts (ATCC no. CRL-1772) used to harbor parasites were
cultivated in Dulbecco’s Modified Eagle’s Medium containing 4.5 g/L
glucose (DMEM H-21), supplemented with 5% FBS, 25 mM HEPES,
2
mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL
74
streptomycin. T. cruzi CA-I/72 trypomastigotes were obtained
from infected-culture supernatants after 4−7 days of infection.
Cultures were maintained at 37 °C with 5% CO . Trypomastigotes
2
and C2C12 cells concentration was determined using a Neubauer
hemocytometer.
5
intraperitoneally, 10 trypomastigotes per mouse. Three days post
The assay was performed in triplicate. Sterile, black 384-well plates
infection, mice were anesthetized by inhalation of isofluorane
with clear-bottom wells (Greiner Bio-One) were seeded with mouse
(controlled flow of 1.5% of isofluorane in air was administered
M
dx.doi.org/10.1021/jm500448u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
through a nose cone via gas anesthesia system). Mice were injected ip
with 150 mg/kg D-luciferin potassium salt (Gold Biotechnology)
dissolved in PBS and imaged after 5 min using IVIS Spectrum
preclinical in vivo imaging system (PerkinElmer, Waltham, MA) and
the data acquisition and analysis software LivingImage V4.1
mixing with 20 mM potassium phosphate pH 7.5, 10% glycerol, 1 mM
DTT, 0.5 mM EDTA, and 300 mM NaCl, supplemented with
equimolar inhibitor. Crystallization conditions were determined using
commercial high-throughput screening kits available in deep-well
format (Hampton Research, Aliso Viejo, CA), a nanoliter drop-setting
Mosquito robot (TTP LabTech, Cambridge, MA) operating with 96-
well plates, and a hanging drop crystallization protocol. Crystals were
further optimized in 96-well plates for diffraction data collection and
harvested directly from the 200 nL drops. Prior to data collection,
crystals were cryoprotected by plunging them into a drop of reservoir
solution supplemented with 20% ethylene glycol, then flash frozen in
liquid nitrogen.
(
PerkinElmer, Waltham, MA). Only mice with detectable lumines-
cence at day 3 postinfection were used for treatment. Compound
potency was evaluated following intaperitoneal (ip) or oral (po)
administration in groups of five mice.
Intraperitoneally, treatment with compounds continued at 40 mg/
kg, bid, for four consecutive days. The compounds were administered
at 8 mg/mL in 100 μL of 60% DMSO in saline (NaCl 0.9%) per dose.
Two control groups included untreated mice, which received 60%
DMSO in saline (NaCl 0.9%), and the positive control group, which
received 20 mg/kg posaconazole, both ip, bid. Orally, compounds
were administered as suspension in 20% 2-hydroxypropyl-β-cyclo-
dextrin (HPβCD) (VWR International) or as solution in Kolliphor HS
Diffraction data were collected at 100−110 K at beamline 8.3.1,
Advanced Light Source, Lawrence Berkeley National Laboratory, USA.
Data indexing, integration, and scaling were conducted using
7
8
MOSFLM and the programs implemented in the ELVES software
7
9
suite. The crystal structures were determined by molecular
replacement using diffraction data processed in the corresponding
space groups and atomic coordinates of T. cruzi CYP51 (PDB ID
code: 2WX2) as a search model. The final model was built using
1
5 (Sigma no. 42966), also known as solutol. Only one compound, 7,
was fully soluble in HPβCD, while all dissolved in 20% solutol upon
overnight incubation. Specified doses were administered bid for four
consecutive days. Two control groups included untreated mice, which
received 20% HPβCD or solutol, and the positive control group, which
received benznidazole 50 mg/kg, both bid by oral gavage. After 4 days
of treatment, mice were imaged again as described above. The absolute
8
0
COOT and refinement was performed by using REFMAC5
8
1,82
software.
Data collection and refinement statistics are shown in
Supporting Information, Table S3.
2
numbers of measured photons/s/cm were averaged between all five
ASSOCIATED CONTENT
Supporting Information
■
mice in each group and compared directly between compound-treated
mice and the control groups. Two-tailed paired Student t test was used
to assess statistical significance between luminescence values from
vehicle-treated and compound-treated groups at day 7 postinfection;
values are statistically significant when p ≤ 0.05.
Single Dose PK and Tissue Distribution. Compounds were
dosed at 50 mg/kg in eight-week-old female Swiss Webster albino
mice via oral gavage. All compounds were formulated to a
concentration of 10 mg/mL in 20% solutol/80% water. Nine mice
were dosed and n = 3 plasma samples were collected at approximately
*
S
Synthetic procedures and tabulated spectroscopic data for
synthetic intermediates, tissue distribution data, dose−response
curves, UV−vis titration curves, lipid profiles by GC-MS
chromatograms and crystallographic statistics. This material is
available free of charge via the Internet at http://pubs.acs.org.
Accession Codes
The atomic coordinates and structure factors (PDB ID codes
4
C0C, 4UQH, and 4BMM) have been deposited in the Protein
0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Tissue samples were collected from
Data Bank, Research Collaboratory for Structural Bioinfor-
matics, Rutgers University, New Brunswick, NJ (http://www.
rcsb.org/).
three mice two and 8 h post dose. Plasma samples were treated with 5
times v/v acetonitrile to precipitate protein and filtered through a 0.2
μm filter prior to analysis by LC-MS/MS using an ABSciex 5500.
Tissue samples were placed in PBS (1:4 w/v) and disrupted using a
probe tip sonicator prior to precipitating protein with acetonitrile and
LC-MS/MS analysis.
AUTHOR INFORMATION
Corresponding Authors
For L.M.P.: phone, (415) 514-1381; fax, (415) 502-8193; E-
■
*
Sterol Profiling of Intracellular T. cruzi Amastigotes. Sterol
profiling was performed on whole-cell lipid extracts, prepared as
51
mail, larissa.podust@ucsf.edu; address, Department of Pathol-
ogy, University of California, San Francisco, California, 94158,
United States.
*For W.R.R: phone, (561) 228-2450; fax, (561) 228-3052; E-
mail, roush@scripps.edu; address, Department of Chemistry,
Scripps Florida, Jupiter, Florida 33458, United States.
described previously. Posaconazole (100 nM) was used as a positive
control and benznidazole (5 μM) as a negative control. Compounds 3
and 12 were tested at 100 nM. Briefly, C2C12 cultures infected with T.
cruzi (CAI/72 strain) for 96 h and treated with compounds for 24 h
were detached and submitted to lipid extraction with chloroform/
methanol, chloroform, and acetonitrile, each step followed by several
rounds of washes with water to extract polar molecules. The organic
layer was then dried under nitrogen gas and subsequently treated with
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
75 μL of N,N-bis(trimethylsilyl)-2,2,2-trifluoroacetamide (BSTFA) for
2
h at 37 °C to facilitate chemical derivatization with trimethylsilyl
(
TMS) groups (BSTFA, Sigma-Aldrich). The TMS-derivatized lipid
mixture was analyzed by injecting 3 μL directly into an Agilent
HP5790 gas chromatography system outfitted with a DB5-MS
analytical column (30 m, 0.25 mm i.d., 0.33 μm film thickness,
Agilent) coupled to a mass selective detector. The lipids were
separated on the analytical column using a temperature profile that
begins at 200 °C for 1 min, increases by 15 °C/min up to 300 °C, and
then holds at 300 °C for 20 min. The inlet temperatures of the GC
and the MSD were held at 250 and 300 °C, respectively. The mass
spectrometer scanned from m/z 50−750 during the course of analysis.
X-ray Structure Analysis. To analyze the inhibitor binding mode,
recombinant T. cruzi CYP51, modified by replacing the first 31
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Barbara Burleigh, Harvard School of Public
Health, and Dr. Ana Rodriguez, New York University, for
kindly providing T. cruzi Y luc strain, Dr. Julio Urbina for
fruitful discussions and expert advice, Potter Wickware for
proof reading of the manuscript, the staff members of beamline
8.3.1, James Holton, George Meigs, and Jane Tanamachi, at the
Advanced Light Source at Lawrence Berkeley National
Laboratory, for assistance with data collection. We also thank
the Ortiz de Montellano Laboratory at UCSF for use of the
7
7
residues upstream of Pro32 with the fragment MAKKTSSKGKL and
by inserting a His -tag at the C-terminus, was expressed and purified as
6
42
described elsewhere. Concentrated purified protein samples were
stored at −80 °C and diluted prior to crystallization to 0.1 mM by
N
dx.doi.org/10.1021/jm500448u | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
GC-MS instrument and Renata Soares and Liliane Mesquita
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Posaconazole: when and how? The clinician’s view. Mycoses 2011, 55
(
IOC, FIOCRUZ, R.J.) for technical assistance with the
(
2), 110−122.
16) Molina, J.; Martins-Filho, O.; Brener, Z.; Romanha, A. J.;
Loebenberg, D.; Urbina, J. A. Activities of the triazole derivative SCH
6592 (posaconazole) against drug-resistant strains of the protozoan
hepatocyte and cardiomyocyte cultures. This work was
supported by NIH RO1 grant AI095437 (to L.M.P.). C.M.C.
was supported by Conselho Nacional de Desenvolvimento
Cientifico e Tecnologico (CNPq) and FIOCRUZ. J.B.J. was
supported by Dr. Miriam & Sheldon G. Adelson Medical
Research Foundation (APNRR Proteomics Platform,
A120343). The Advanced Light Source is supported by the
Director, Office of Science, Office of Basic Energy Sciences, of
the U.S. Department of Energy under contract no. DE-AC02-
(
5
parasite Trypanosoma (Schizotrypanum) cruzi in immunocompetent
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■
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́
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